We propose to continue our structure-function studies of proteins from hwo groups of plus-strand RNA enveloped viruses: the alphaviruses and flaviviruses. Flaviviruses, a genus within the family Flaviviridae, include yellow fever, dengue. West Nile, and fick-borne encephalifis. We will also carry out studies with the alphaviruses, a genus within the Togaviridae family. Several examples of alphaviruses are Sindbis, Semliki Forest, and Venezuelan equine encephalifis. A structural proteomics approach will be employed to investigate the funcfions of viral proteins and cellular proteins involved in virus replicafion. This approach will entail the use molecular genefics, cell biology, biochemistry and structural techniques to probe the role of virus and host proteins in the virus life cycle and to ulfimately describe this process in atomic detail. This will confinue the active collaborafion between three laboratories from the Structural Biology Group at Purdue and two former members ofthe group, Tim Baker at UCSD and Janet Smith at the University of Michigan. Together with Tim Baker's, Wen Jiang's and Michael Rossmann's laboratories, we will use cryo-electron microscopy (cryo-EM) and image reconstrucfions to examine the structure of virions, their intermediates in entry and fusion, and complexes of anfibodies and receptors. The dynamic properties ofthe virus particles will be explored. We will employ electron tomography and immuno-electron microscopy to determine the spafial and temporal organizafion of virus-induced subcellular structures and will begin to identify viral and host components of these structures. We will collaborate with Janet Smith to carry out structural and biochemical studies on the replicafion proteins from flaviviruses, and will pursue the funcfional identificafion of cellular partners. These studies will be combined with molecular genefic approaches to examine structure-function relafionships in these proteins. The proposed research will advance our knowledge of viral protein structure and funcfion and will be integrated toward an understanding ofthe life cycles of these two groups of viruses. Comparative analyses between members ofthe same genus will provide informafion into the unique nature of specific viruses as well as providing evolufionary insight into protein funcfion and virus origins. RELEVANCE (See instructions): The alphaviruses and flaviviruses comprise a group of insect-transmitted viruses that are responsible for significant human disease. They are emerging pathogens and have been considered as bio-weapons. This project will develop new structural data that will be used to drive genefic, biochemical, and cell biology experiments designed to interrogate viral protein function.